The present invention relates to the field of compounds which are substrates or inhibitors of proteolytic enzymes and to apparatus and methods for identifying substrates or inhibitors for proteolytic enzymes.
Many therapeutically useful drugs act as enzyme inhibitors. In particular, proteolytic enzyme inhibitors have been the focus of much attention in the pharmaceutical industry, because they play a variety of roles in a multitude of biological systems. Their proteolytic activities are related to processes ranging from cell invasion associated with metastatic cancer to evasion of an immune response, as seen in certain parasitic organisms; from nutrition to intracellular signalling to the site-specific proteolysis of viral proteases and eukaryotic hormone-processing enzymes. However, the traditional random screening methods for the identification of lead molecules as inhibitors of proteolytic enzymes are often laborious and time-consuming. Therefore new and efficient methods which can accelerate the drug discovery process are greatly in demand.
We consider that proteases contain an active catalytic site which tends to become increasingly activated as the recognition pockets1 (S1 and S2 etc) and (S1xe2x80x2 and S2xe2x80x2 etc) become better occupied. Therefore, it is important that those parts (P1 and P2 etc) (P1xe2x80x2 and P2xe2x80x2 etc) of the inhibitor that best fit into these pockets are identified as quickly as possible in order to design novel protease inhibitors. Therefore, we have devised a combinatorial method for the rapid identification of these binding motifs which will greatly expedite the synthesis of inhibitors of a variety of proteolytic enzymes such as aspartyl proteases, serine proteases, metallo proteases and cysteinyl proteases.
Proteases of interest include (but are not limited to):
1. Aspartyl proteases, such as renin, HIV, cathepsin D and cathespin E etc.
2. Metalloproteases, such as ECE, gelatinase A and B, collagenases, stromolysins etc.
3. Cysteinyl proteases, such as apopain, ICI, DerPI, cathepsin B, cathepsin K etc.
4. Serine proteases, such as thrombin, factor VIIa, factor Xa, elastase, trypsin.
5. Threonyl proteases, such as proteasome S.
The use of a fluorescence resonance energy transfer (FRET) substrate for the analysis of proteolytic enzyme specificity was first published by Carmel.2 Since then many different quenched fluorogenic substrates for measuring enzyme inhibition have been described in the literature.4-11 These substrates contain a fluorophore, F, in a P position (vide supra), which is quenched by another group, Q, present in a Pxe2x80x2 position (vide supra) and separated from F by the scissile bond. The advantage of the positioning of these residues, F and Q, is that cleavage of a peptide bond occurs between the two natural residues and, therefore, represents a more natural hydrolytic event rather than the cleavage and release of a C-terminal chromophore.
For example, Bratovanova and Petkov12 have synthesised fluorogenic substrates from peptide 4-nitroanilides. N-acylation of peptide 4-nitroanilides with the aminobenzoyl (ABz) group yielded substrates that are internally quenched by the presence of the 4-nitroanilide moiety. Upon hydrolysis of the aminoacyl-4-nitroanilide bond, the highly fluorescent N-ABz group is released attached either to an amino acid or peptide.
Immobilised libraries; where substrates are attached to a polymer or biopolymer support, have also been used for mapping protease binding sites.13 Singh et al. reported recently that enzymatic substrate activity of 38 selected octapeptides attached via a linker to controlled pore glass is predictive of the same activity of similar peptides in solution. However, these results are preliminary and only for a specific example. Therefore, it is not clear whether immobilised substrates attached to polymers can reliably replace soluble substrates in mapping the hindered protease binding sites, especially since the hydrophilic or lipophilic nature of the polymer and the size of the interstices within the polymer are bound to influence the reaction between the enzyme and its substrates.
Mixtures of internally quenched, fluorogenic substrates have also recently been described in which the quencher group, Q, is 2,4-dinitrophenyl (Dnp) and is attached to the P side of the scissile bond, while the fluorogenic group, is N-methyl anthranilic acid (Nma) and is attached to the Pxe2x80x2 side.14 
Examples of other Donor-Acceptor Chromophore Pairs that have been applied to Biological Systems are shown in Table 1.
from: Wu, P. and Brand, L. 1994. Anal. Biochem. 218, 1-13.
The specificity of soluble peptide libraries have been determined.15,16 Berman et al. described16 an HPLC mass spectrometry technique in which 6 mixtures of 128 peptides were synthesised which were N-terminally labelled with the Dnp group in order to allow UV monitoring on the HPLC. The disadvantage of this approach is that each assay mixture has to be individually analysed, because no fluorogenic substrate is revealed, and that the effective concentration of each separate component is limited by the size of the mixture because of overall solubility factors. Drevin et al.17 have suggested the use of individually synthesised fluorogenic substrates for the determination of enzyme activity using a chromophore which chelates lanthanide ions. Garmann and Phillips have suggested the use of FRET substrates in which the fluorogenic and quencher moieties are attached via thiol or amino functional groups after the peptide has been synthesised, but this has the disadvantage that they are not in library form and that these functional amino and thiol groups need to be selectively revealed after the peptide has been synthesised. Wang et al. have suggested the use of the EDANS and DABCYL fluorescor and quencher pairing for the individual synthesis of substrates for proteolytic enzymes.
The above methods which have used FRET techniques for the mapping of the active site around a specific protease suffer from one or more of the following disadvantages:
i. because of general aqueous insolubility they do not produce mixtures of compounds in a form suitable for high throughput screening in aqueous solution.
ii. the derivatised compounds cannot be prepared in combinatorial library form using solid phase techniques.
iii. the mixtures which have been used8,9 were not self-decoding, and needed time-consuming deconvolutive resynthesis for identification of the active molecules.